ABSTRACT Infection with the hepatitis B virus (HBV) is still a major global health problem with an estimated 6% of the world’s population chronically infected with the virus. Chronic infection with HBV subgenotype A1, which is hyperendemic to southern Africa, is associated with a particularly high incidence of liver cancer and cirrhosis. Understanding HBV replication and developing effective HBV treatment to prevent liver cancer remain important medical priorities. Although there is a preventative vaccine for HBV, efficacy of currently available treatment of established infection is limited. Exploiting the RNA interference (RNAi) pathway through the use of small interfering (siRNA) and short hairpin RNA (shRNA) is an attractive new approach for the development gene therapies against HBV infection. Our laboratory has designed and demonstrated the efficacy both in vitro and in vivo of several shRNAs designed to target the X open reading frame (ORF) of HBV. Thus, the objective of this study was to construct a replication competent plasmid vector of the A1 subgenotype, a reporter plasmid vector of HBV and to assess the efficacy of RNAi effecters against these vectors both in vitro and in vivo. The first HBV replication competent vector, pCR-HBVA1 1.3x, containing the sequence of an HBV subgenotype A1 isolate, was successfully constructed by generating a greater than genome length sequence of HBV, that starts just upstream of endogenous HBV basic core promoter (BCP) and ends just downstream of the unique HBV polyadenylation (pA) site. Human hepatoma (Huh7) cells transfected with this plasmid secreted HBV surface antigen (HBsAg) into Abstract viii culture supernatants. In the murine hydrodynamic injection model of HBV replication, serum HBsAg, hepatitis B e antigen (HBeAg) and viral particle levels as well as relative surface and core mRNA levels were shown to be significantly elevated as compared to mock-injected mice. The second HBV vector, pCH-FLuc, was successfully generated by replacing the surface ORF with the sequence encoding Firefly Luciferase. The ability of pCH-FLuc to express Firefly Luciferase was demonstrated in a liver cell line (Huh7 cells). Co-transfection of the reporter plasmid, pCH-FLuc, with shRNAs targeted to HBV caused a significant reduction in Luciferase expression. Co-transfection/injection of the pCR-HBVa1 1.3x with shRNAs caused significant inhibition in the level of viral antigens (HBsAg, HBeAg and hepatitis B core antigen (HBcAg) as well as relative surface and core mRNA levels. This was observed both in vitro and in vivo. Our results demonstrate the potential this model allows for the study of HBV replication as well as the assessment of potential therapeutic strategies in a regionally significant subgenotype of HBV.

M.Sc. (Epidemiology), Faculty of Health Sciences, University of the Witwatersrand, 2011 / Background and Objectives The advent of Highly Active Antiretroviral Therapy (HAART) has resulted in a significant reduction in HIV/AIDS related morbidity and mortality in sub-Saharan Africa. However, toxicities due to HAART continue to pose challenges to the success of different regimens. Severe hepatotoxicity is one of the significant adverse events occurring in patients on HAART. Information on the incidence and risk factors for severe hepatotoxicity in cohorts from resource poor settings is limited. It is against this background that we undertook the study to determine the incidence and explore factors associated with severe hepatotoxicity following HAART initiation in a South African cohort. Materials and Methods Secondary data analysis of a prospective cohort 9764 HIV-infected adult patients initiated on HAART at the Themba Lethu clinic antiretroviral rollout facility in Johannesburg, South Africa between 1st April 2004 and 30th June 2009 was conducted. Severe hepatotoxicity cases were identified within the first 12 months of initiating HAART as grade 3 or 4 elevation in baseline ALT levels. The incidence rate of severe hepatotoxicity was calculated and potential socio-demographic and clinical predictors were explored using Cox proportional hazard regression modelling. Results At baseline, 91.8% of patients were commenced on an efavirenz-based regimen while only 8.2% were on a nevirapine-based regimen. The median CD4 count at v initiation of HAART for this cohort was 80 cells/ mm3, a figure lower than the Department of Health (DoH) CD4 cut off for initiating HAART of 200 cells/ mm3. The overall incidence rate of severe hepatotoxicity was 10.7 (95% CI: 8.7 – 13.1) cases per 1000 p-yrs of follow-up. The period with the highest risk of severe hepatotoxicity was within 2 months of initiating HAART. Incidence of severe hepatotoxicity was 21.1(95% CI: 12.7 – 34.9) cases per 1000 p-yrs among patients on a nevirapine-based regimen and 9.7 (95% CI: 7.8 – 12.1) cases per 1000 p-yrs in those on an efavirenz-based one. The hazard for severe hepatotoxicity within the first year of initiating HAART was 2.17 times higher in individuals on a nevirapine-based regimen compared to those on an efavirenz-based regimen after adjusting for baseline ALT, baseline CD4, age and gender (HR = 2.17; 95%CI = 1.18 – 3.97; p = 0.013). Though imprecise, the estimate for baseline ALT category suggested an increased risk for severe hepatotoxicity in individuals with a baseline ALT more than 40 I.U/L compared to those with a baseline ALT of less than 40 I.U/L (HR = 1.63; 95%CI = 1.00 – 2.67; p = 0.050). Conclusion The results of the study suggest that severe hepatotoxicity following initiation of HAART in this cohort is low compared to other previously studied cohorts. The high incidence rate of severe hepatotoxicity in the first two months of initiating HAART necessitates the need for more frequent and careful monitoring of ALT levels early during therapy. Patients on a nevirapine-based regimen have a higher risk of developing severe hepatotoxicity when compared to their counterparts on an efavirenz-based regimen, a result consistent with findings from previous studies.

Hepatitis B virus (HBV) is hyperendemic to southern Africa and parts of Asia where it is a major cause of serious liver disease. Licensed antivirals for chronically infected individuals are only partially effective and approximately one million deaths occur annually as a result ofpersistent infection with the virus. Although RNA interference (RNAi) based gene silencingof HBV has been successfully demonstrated, difficulties with delivery of anti-HBV RNAieffectors remains an obstacle to their clinical use. Recombinant adenoviruses (Ads), amongst the most efficient hepatotropic gene vectors following systemic administration, have been successfully used to deliver expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate andadaptive immunity.To attenuate these effects we used polyethylene glycol (PEG) to modify first generation recombinant Ad (FG Ad) vectors that express an anti-HBV short hairpin (shRNA) sequence. Efficient hepatocyte transduction occurred and expressed shRNAs were processed to generate intended HBV-targeting guides. Inhibition of HBV replication was achieved after intravenous administration of PEGylated or native recombinant first generation Ads (FG Ads) to HBV transgenic mice. Circulating HBV viral particle equivalents (VPEs) remained low for 3 weeks and began to increase after 5 weeks. A second dose of PEGylated anti-HBV Ad caused a less sustained decrease in circulating VPEs, but no silencing after a second dose was observed in animals treated with unmodified vector. Release of inflammatory cytokines was elevated in animals receiving unmodified vectors and only a modest increase in monocyte chemotactic protein-1 (MCP-1) was observed in mice that received a second dose of PEG Abstract Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts. To address concerns about the transient nature of transgene expression by FG Ads resulting from immunostimulation, third generation helper-dependent (HD Ad) were utilised to delivered anti-HBV RNAi effectors. Seven days after intravenous administration of infectious HD Ads to HBV transgenic mice, 80-90% of hepatocytes were transduced and markers of HBV replication were decreased by approximately 95% which was sustained for 8 weeks. HD Ad-induced release of proinflammatory cytokines was minimal in preparations that were enriched with infectious particles. PEGylated HD Ad vectors caused similar anti- HBV effects and may be useful to evade interaction with vector-sequestrating receptors and further attenuate immunostimulation. Collectively these observations indicate that PEG modification of Ads and the use of HD Ads may have utility for delivery of therapeutic HBVsilencing sequences. Future work will focus on improving strategies to avoid immune detection and utilisation of HD Ad vectors for other HBV targeting sequences.

Thesis (Ph.D.)--University of the Witwatersrand, Faculty of Health Sciences, 2013.

Law Man Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 123-132). / Abstracts in English and Chinese; appendix in Chinese. / Precis --- p.vi / 摘要 --- p.viii / Captions for Tables --- p.x / Caption for Figure --- p.xii / Abbreviations --- p.xiii / Chapter Part 1 --- Literature Review --- p.0 / Chapter Chapter 1 --- Aims and Hypothesis --- p.1 / Chapter 1.1 --- Aims --- p.3 / Chapter 1.2 --- Hypothesis --- p.4 / Chapter Chapter 2 --- Epidemiology of HBV Infection --- p.5 / Chapter 2.1 --- Global and Local Epidemiology --- p.6 / Chapter 2.2 --- Modes of Transmission --- p.8 / Chapter 2.2.1 --- Perinatal Tansmission --- p.8 / Chapter 2.2.2 --- Percutaneous Transmission --- p.8 / Chapter 2.2.3 --- Sexual Transmission --- p.8 / Chapter 2.2.4 --- Healthcare Setting and Transplantation --- p.8 / Chapter 2.2.5 --- Transfusion --- p.9 / Chapter 2.2.6 --- Other --- p.9 / Chapter Chapter 3 --- Popularity of TCM --- p.10 / Chapter 3.1 --- Traditional Chinese Medicine Use --- p.11 / Chapter 2.2 --- Regulation on TCM Practice --- p.12 / Chapter Chapter 4 --- Philosophy of TCM on HBV Infection --- p.13 / Chapter 4.1 --- Basic Principles --- p.14 / Chapter 4.1.1 --- Yin-Yang Theory --- p.14 / Chapter 4.1.2 --- Five Elements Theory --- p.14 / Chapter 4.1.3 --- Zang Fu Theory --- p.14 / Chapter 4.2 --- Pathogenesis --- p.16 / Chapter 4.3 --- Diagnosis --- p.17 / Chapter 4.4 --- Treatment --- p.20 / Chapter Chapter 5 --- Western Medicine --- p.23 / Chapter 5.1 --- Natural History of HBV Infection --- p.24 / Chapter 5.1.1 --- Replicative Phase: Immune Tolerance --- p.24 / Chapter 5.1.2 --- Replicative Phase: Immune Clearance --- p.24 / Chapter 5.1.3 --- Noreplicating (Low-Replication) Phase --- p.25 / Chapter 5.2 --- Diagnostic Tests for HBV Infection --- p.26 / Chapter 5.2.1 --- Serologic Assays --- p.26 / Chapter 5.2.2 --- Serum Enzymes --- p.26 / Chapter 5.2.3 --- HBV DNA Assays --- p.27 / Chapter Chapter 6 --- Health-related Quality of Life --- p.29 / Chapter 6.1 --- Principle and Definition --- p.30 / Chapter 6.2 --- Assessment --- p.31 / Chapter 6.2.1 --- Generic Instrument --- p.31 / Chapter 6.2.2 --- Disease-Specific Instrument --- p.32 / Chapter Part 2 --- Studies & Results --- p.33 / Chapter Chapter 7 --- Research Methodology --- p.34 / Chapter 7.1 --- Study 1: Hospital-Based Surveys on Chronic Hepatitis B Patients: TCM Use --- p.35 / Chapter 7.1.1 --- Patients --- p.35 / Chapter 7.1.2 --- Survey Instrument & Logistic --- p.35 / Chapter 7.1.4 --- Statistical Analysis --- p.36 / Chapter 7.1.4 --- Sample Size Justification --- p.36 / Chapter 7.2 --- Study 2: Hospital-Based Survey on Chronic Hepatitis B Patients: HRQoL & Psychiatric Involvement --- p.38 / Chapter 7.2.1 --- Patients --- p.38 / Chapter 7.2.2 --- Survey Instrument and Logistic --- p.38 / Chapter 7.2.3 --- Statistical Analysis --- p.39 / Chapter 7.3 --- Study 3: Population-Based Survey on Chinese Medicine Practitioners (CMPs): Practice Behavior & Knowledge Assessment --- p.41 / Chapter 7.3.1 --- Study Population --- p.41 / Chapter 7.3.2 --- Survey Instrument --- p.41 / Chapter 7.3.3 --- Statistical Analysis & Knowledge Assessment --- p.42 / Chapter 7.4 --- Study 4: TCM Consultation Agreement --- p.44 / Chapter 7.4.1 --- Patients --- p.44 / Chapter 7.4.2 --- Chinese Medicine Practitioners --- p.44 / Chapter 7.4.3 --- Questionnaire --- p.44 / Chapter 7.4.4 --- Study Design --- p.45 / Chapter 7.4.5 --- Sample Size Estimation --- p.45 / Chapter 7.4.6 --- Data Analysis --- p.45 / Chapter 7.5 --- "Study 5: TCM Interpretation of Laboratory, Imaging & HRQoL Assessment" --- p.47 / Chapter 7.5.1 --- Patients --- p.47 / Chapter 7.5.2 --- HRQoL Assessment --- p.47 / Chapter 7.5.3 --- Statistical Analysis --- p.47 / Chapter Chapter 8 --- TCM Use on Chronic Hepatitis B Patients --- p.48 / Chapter 8.1 --- Patient Characteristics --- p.49 / Chapter 8.2 --- Health-Seeking Behavior of TCM Users --- p.51 / Chapter 8.3 --- Determinants of TCM Use --- p.53 / Chapter 8.4 --- Common Herbal Ingredients Used --- p.56 / Chapter Chapter 9 --- Impacts of HBV Infection on Patients --- p.58 / Chapter 9.1 --- Patient Socio-Demographic and Clinical Characteristics --- p.59 / Chapter 9.2 --- Patient Symptoms and Anxiety /Depression --- p.61 / Chapter 9.3 --- HRQoL & its Determinants --- p.63 / Chapter 9.3.1 --- Physical Aspect of HRQoL --- p.63 / Chapter 9.3.2 --- Mental Aspect of HRQoL --- p.67 / Chapter Chapter 10 --- Chinese Medicine Practitioners' (CMP) Practice --- p.71 / Chapter 10.1 --- CMPs Demographics and Training --- p.72 / Chapter 10.2 --- Practice Behavior --- p.75 / Chapter 10.3 --- Diagnostic and Therapeutic Approach --- p.76 / Chapter 10.4 --- Professional Knowledge Assessment --- p.79 / Chapter 10.5 --- Determinants of Diagnostic Approach --- p.82 / Chapter Chapter 11 --- Agreement of TCM Diagnosis among Different CMPs --- p.85 / Chapter 11.1 --- Patients Characteristics & Disease Severity --- p.86 / Chapter 11.2 --- Agreement of TCM Diagnosis & Treatment --- p.88 / Chapter Chapter 12 --- Interpretation of TCM diagnosis --- p.93 / Chapter 12.1 --- Patients --- p.94 / Chapter 12.2 --- Clinical Characteristics --- p.96 / Chapter 12.3 --- HRQoL Assessment --- p.98 / Chapter Part 3 --- Discussion & Conclusions --- p.100 / Chapter Chapter 13 --- Discussion --- p.101 / Chapter 13.1 --- TCM Popularity and its Practice --- p.102 / Chapter 13.2 --- Impact of TCM Symptomatology & Anxiety /Depression on HRQoL --- p.107 / Chapter 13.3 --- TCM Diagnosis Consistency --- p.111 / Chapter 13.4 --- Association of TCM Diagnosis with Western Medicine --- p.114 / Chapter 13.5 --- Limitations of the Study --- p.117 / Chapter Chapter 14 --- Conclusions --- p.120 / Reference --- p.122 / List of Publications of My Work Used in This Thesis --- p.133 / Acknowledgement --- p.136 / Appendix --- p.138

Globally, it is estimated that 240 million people are infected with chronic viral hepatitis B and in excess of 185 million people with chronic hepatitis C. The burden of disease from hepatitis is concentrated in developing countries where transmission of HBV occurs predominantly from mother to child (vertical transmission) and transmission of HCV through unsafe medical procedures and the transfusion of unscreened blood products. Global patterns of migration favour the movement of individuals from countries with medium or high risk prevalence of chronic viral hepatitis to countries with traditionally low prevalence among their indigenous populations, including the United Kingdom (UK). In excess of 3.2% of the global population are international migrants, posing important implications for healthcare systems in host nations. It is predicted that up to 7 million first and second generation immigrants, originating from high prevalence countries for viral hepatitis now reside permanently in the UK. However, as a result of deficiencies in screening initiatives, the prevalence and associated burden of these diseases in these high-risk populations residing in the UK is yet to be determined. In order to establish the feasibility of inviting first and second generation immigrant populations to participate in viral hepatitis testing in primary care, as well to determine the prevalence and demography of viral hepatitis in four areas of the UK, a randomised controlled cross sectional cluster trial was conducted. In HepFree clinical computer systems in general practice surgeries were interrogated to identify the target population that was then approached using a variety of different invitations to determine the most appropriate method for engaging this population. The outcomes of viral hepatitis testing from practices in one area of the UK are described in this thesis. Despite multiple challenges encountered both in engaging practices and individuals in trial participation, results of this investigation suggest that if it is found to be cost effective, then viral hepatitis screening is feasible and the burden of disease in the UK is concentrated in first generation immigrants.

OBJETIVO: Evaluar el conocimiento del impacto en la morbilidad y mortalidad de la hepatitis B, enfermedad por Haemophilus influenzae tipo b, parotiditis y rubéola y sus respectivas vacunas en una población de médicos de la región Chavín. DISEÑO: Estudio Observacional Descriptivo MATERIAL Y METODO: Se aplicó un cuestionario-encuesta transversal en los meses de mayo y junio del 2,003. Se incluyeron a 125 médicos generales de la región Chavín, los cuales fueron distribuidos en tres grupos de acuerdo a la antigüedad de egreso de la universidad RESULTADOS: Un 30.0% respondieron correctamente con respecto a la enfermedad por el virus de la hepatitis B (13.8% correspondió a los médicos egresados desde hace menos de un año, p = 0.008) y un 32.4% con respecto a su vacuna (la mayoría correspondió a médicos egresados desde hace menos de un año, p < 0.001). Un 57.6% respondieron correctamente con respecto a la enfermedad por el Haemophilus influenzae tipo B (21.6% correspondió a los médicos egresados desde hace menos de un año, p = 0.03) y un 15.8% con respecto a su vacuna (6.6% correspondió a médicos egresados desde hace menos de un año, p = 0.094). Un 39.6% respondieron correctamente con respecto a la enfermedad paperas-rubéola-sarampión (18.6% correspondió a los médicos egresados desde hace menos de un año, p < 0.001)) y un 40.4% con respecto a su vacuna (17.8% correspondió a médicos egresados desde hace menos de un año, p = 0.004). Un 87.2% refirió a la inaccesibilidad económica como principal obstáculo para indicar estas vacunas, un 72% no informan a los padres, los que si lo hacen sólo emplean entre 1 a 5 minutos de la consulta médica, 67.2% responden conocer alguna asociación vacunal y 60% se colocaron alguna dosis. CONCLUSIONES: Existe un bajo índice de conocimientos con respecto a las enfermedades en estudio, aunque significativamente mayor en relación a sus vacunas, siendo mejor en el grupo de médicos egresados desde hace menos de un año de la universidad. No se conocen bien las indicaciones, contraindicaciones, cadena de frío e impacto de sus respectivas vacunas en la población infantil. El costo es una de las principales barreras encontradas y no se brinda información a los padres sobre estas vacunas. PALABRAS CLAVE: inmunizaciones, conocimientos, médicos, prácticas / Tesis de segunda especialidad

The hepatitis C virus (HCV) currently infects 3% of the world???s population, with chronic infection in 50-80% of exposed individuals. A small subset of individuals who are exposed to HCV do not develop anti-HCV antibodies, persistent viraemia or chronic hepatitis despite generating HCV-specific CD4+ and CD8+ T cells. These individuals are believed to develop an immune response which rapidly clears viraemia prior to the induction of an antibody response. Circumstantial evidence supports the likelihood that some of these individuals may generate these same responses and outcomes on repeated occasions of HCV infection. HCV-specific cellular immune responses in seronegative subjects have been the subject of only limited prior study, in part due to the lack of appropriate recombinant antigens and assay systems. Therefore, this thesis described the development and validation of an interferon-? (IFN-?) ELISPOT assay using overlapping peptides (n=441). Using this assay, HCV-specific cellular immune responses were detected in 5/10 (50%) of chronically infected subjects. Responses were identified more frequently, and were directed against more regions of the HCV genome, than with traditional assay systems. This IFN-? ELISPOT assay, a comparable interleukin (IL)-2 ELISPOT assay, and a multiplex in vitro cytokine production assay were then used to evaluate HCV-specific cellular immune responses in three cohorts of seronegative subjects at high-risk of exposure to HCV ??? babies born to infected mothers, multiply-transfused subjects with thalassaemia, and high risk injecting drug users. Cellular immune responses were evaluated in 23 infants born to HCV-antibody positive women. Responses were not detected in infants born to HCV-PCR negative mothers. IFN-? production was detected in 1/11 infants born to viraemic mothers using the ELISPOT assay, with cytokine production observed in an additional 3/5 infants studied using the in vitro cytokine production assay. HCV-specific cellular immune responses were assessed in a cohort of multiply transfused subjects with thalassaemia using assays for cytotoxic T lymphocyte activity, IFN-? and IL-2 ELISPOT, as well as lymphocyte proliferation and in vitro cytokine production. Responses were detected in 6/13 chronically infected subjects (46%), 4/7 subjects who had cleared infection (71%), and 14/17 seronegative subjects (82%). The seronegative subjects had responses which were broader and higher in magnitude than those with chronic HCV infection, although lower and narrower than in subjects who had cleared prior HCV infection. IFN-? and IL-2 ELISPOT assays, in additional to in vitro cytokine production assays, were performed on 41 injecting drug users (IDUs), with responses detected in 6 (15%). Seronegative IDUs with HCV-specific cellular immune responses had been injecting for a mean of 7.7 years, and reported multiple risk factors for exposure to HCV. The combined data from these three cohorts indicate that the HCV-specific cellular immune responses detected in seronegative subjects were generally broad in specificity. Cytokine production was generally Th1-biased, a pattern which has previously been associated with an increased likelihood of clearance in primary infection. The findings also suggest that responses can be maintained for decades after exposure, and may provide protection against repeated exposures. In summary, cellular immunity against HCV is evident in some seronegative high risk subjects, suggesting that the cellular immune responses may efficiently facilitate viral clearance. Understanding the mechanisms of this immune response pattern will allow better understanding of the host response to HCV and may provide key insights into vaccine design.

Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers.<p> Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported.<p> To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection.<p> Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-á, LPS and a maturation cocktail (MC) consisting of IL-1â, IL-6, TNF-á, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-á or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-ã secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.<p> The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients.

Dendritic cells (DCs) constitute a large family of immune cells with a dendritic morphology and a critical role in all aspects of an immune response and immune regulation, from immunogenicity to tolerance. One of the important characteristic of DCs is maturation, during which DCs undergo significant changes in their phenotypic and functional properties and change from phagocytic cells to highly efficient antigen presenting cells (APCs). Dendritic cells have recently been at the centre of attention as a promising tool in treatment or control of cancer and infectious diseases. Accordingly, DCs have been generated, matured, and loaded with tumor-associated or microbial antigens ex vivo, to be subsequently used as therapeutic tools or vaccine carriers.<p> Hepatitis C virus (HCV) is a hepatotropic virus, which infects the liver in humans and results in a chronic infection in most cases. The persistent infection of the liver eventually results in cirrhosis and/or hepatocellular carcinoma in 15-20 years. Chronic hepatitis C (CHC) has recently become a serious health concern and the leading cause of liver transplantation. The mechanism of persistence of the virus is not clear yet, but as a Th1-type immune response is strongly correlated with elimination of HCV in vivo, it is evident that insufficient cellular immunity is a contributing factor. Non-cytopathic viruses such as HCV may infect immune cells to modify and evade a protective immune response. Dendritic cells, which are the most potent APCs, and uniquely capable of initiating a primary immune response, have been considered as a target for HCV. Inhibition of DC maturation by HCV has been suggested as a potential contributing factor in immune evasion; however, this issue remains controversial as many contradictory results have been reported.<p> To investigate this contention, we initially planned to evaluate the effects of HCV on DCs of CHC patients; however, due to limited access to patients blood, we instead elected to examine the effects of HCV genes products on in vitro generated DCs from healthy volunteers. Specific attention was paid to the generation, maturation, and transfection of DCs in vitro, as variability in procedures might have been responsible for the controversial reports. Viral vectors have generally been used to transfect DCs; however, a vector and HCV genes might have synergistic effects on DC maturation. Thus, our first objective was to develop an efficient non-viral transfection method while retaining high viability of the DCs, as previous efforts in this regard resulted either in low efficiency or in low viability of DCs after transfection. In order to improve the viability of DCs after transfection, we established a new method for fast generation of monocyte-derived DCs (Mo-DCs) in two to three days. By performing a comprehensive study on transfection reagents, electroporation, and nucleofection with DNA or in vitro transcribed (IVT) RNA, we successfully established a new, highly efficient non-viral method for transfection of DCs with long-term viability. This method is based on the use of the X1 program of a nucleofection device with IVT RNA and results in high transfection efficiency of 93%, with 75% viability of DCs 72 h after transfection.<p> Subsequently, we performed a comprehensive study on the effects of different maturation methods on the phenotype, function and gene expression profile of DCs. Three commonly used treatments, TNF-á, LPS and a maturation cocktail (MC) consisting of IL-1â, IL-6, TNF-á, and prostaglandin E2 (PGE2) were compared. Our results showed that there is a significant difference in the level of maturity between these treatments, and MC generated more functionally competent mDCs than TNF-á or LPS. In addition, MC induced Th1-promoting changes in the transcriptional profile of mDCs. This observation was important, as the presence of PGE2 in MC was previously challenged based on the potential induction of Th2-biased immune responses. However, our results suggest retaining PGE2 in the cocktail because of the fact that MC generated highly competent and functional mDCs with a Th1-promoting transcriptional profile. Finally, Mo-DCs were transfected with IVT HCV RNAs, individually or in combination. While HCV genes had no inhibitory effect on DC maturation, transfection of DCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of DCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison to NS3 or GFP RNA treatments, 42 of which are involved in DC maturation. The effects of core on maturation of DCs were further confirmed by a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-ã secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.<p> The use of a non-viral method of transfection combined with confirmed transcriptional profiles of DCs in this study may make these results conclusive for in vitro generated DCs from healthy volunteers. However, further investigations are required to confirm the effects on DCs from CHC patients.